Downloaded from https://journals.lww.com/jtrauma by BhDMf5ePHKav1zEoum1tQfN4a+kJLhEZgbsIHo4XMi0hCywCX1AWnYQp/IlQrHD3n4/ufkX3x2fNokYq91MxH/qukLsurVt4psWJZrttSumP18ldoPnO1w== on 07/14/2018 Alterations in the human proteome following administration of valproic acid Patrick E. Georgoff, MD, Ihab Halaweish, MD, Vahagn C. Nikolian, MD, Gerald A. Higgins, MD, PhD, Tess Bonham, MS, Celia Tafatia, Henriette Remmer, PhD, Rajasree Menon, PhD, Baoling Liu, MD, Yongqing Li, MD, PhD, and Hasan B. Alam, MD, Ann Arbor, Michigan BACKGROUND: High doses of the histone deacetylase inhibitor valproic acid (VPA, 150–400 mg/kg) improve outcomes in animal models of lethal insults. We are conducting a US Food and Drug Administration–approved Phase I, double-blind, placebo-controlled trial to eval- uate the safety and tolerability of ascending doses of VPA in human volunteers. We hypothesized that VPA would induce signif- icant changes in the proteome of healthy humans when given at doses lower than those used in prior animal studies. METHODS: Peripheral blood mononuclear cells were obtained from three healthy subjects randomized to receive VPA (120 mg/kg over 1 hour) at baseline and at 4 and 8 hours following infusion. Detailed proteomic analysis was performed using 1D gel electrophoresis, liquid chromatography, and mass spectrometry. Proteins with differential expression were chosen for functional annotation and pathway analysis using Ingenuity Pathway Analysis (Qiagen GmbH, Hilden, Germany) and Panther Gene Ontology. RESULTS: A total of 3,074 unique proteins were identified. The average number of proteins identified per sample was 1,716 ± 459. There were a total of 140 unique differentially expressed proteins (p < 0.05). There was a minor and inconsistent increase in histone and nonhistone protein acetylation. Functional annotation showed significant enrichment of apoptosis (p = 3.5E-43), cell death (p = 9.9E-72), proliferation of cells (p = 1.6E-40), dementia (p = 9.6E-40), amyloidosis (p = 6.3E-38), fatty acid metabolism (p = 4.6E-76), quantity of steroid (p = 4.2E-75), and cell movement (p = 1.9E-64). CONCLUSIONS: Valproic acid induces significant changes to the proteome of healthy humans when given at a dose of 120 mg/kg. It alters the ex- pression of key proteins and pathways, including those related to cell survival, without significant modification of protein acety- lation. In the next part of the ongoing Phase I trial, we will study the effects of VPA on trauma patients in hemorrhagic shock. (J Trauma Acute Care Surg. 2016;81: 1020–1027. Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.) LEVEL OF EVIDENCE: Therapeutic study, level V. KEY WORDS: Cell regulation; histone deacetylase inhibitors; pathways; proteomics; valproic acid. H emorrhagic shock results in poor tissue perfusion and end- organ dysfunction. The treatment of hemorrhagic shock focuses on rapid control of bleeding and aggressive fluid resus- citation. Historically, resuscitation was carried out with crystal- loid fluids. However, this has been shown to worsen outcomes and exacerbate cellular dysfunction. 1,2 While blood products are now recognized as the preferred resuscitative fluid for pa- tients in hemorrhagic shock, 3 their administration is limited by logistical constraints, including limited supply, short half-life, and the need for refrigeration and cross matching. As more than half of hemorrhagic shock deaths occur before arrival to a hospital, 4 a portable, easily administered treatment that can rap- idly ameliorate cellular dysfunction and serve as a bridge to defin- itive care is of great interest. Valproic acid (VPA) is a histone deacetylase (HDAC) in- hibitor specific to histone Classes Ia, Ib, and IIa that was origi- nally approved by the US Food and Drug Administration (FDA) as an antiseizure medication. We have shown that VPA improves survival in otherwise fatal models of hemorrhagic shock and polytrauma in swine and does so in the absence of conventional fluid resuscitation. 5,6 Similar outcomes have been shown in combined models of hemorrhagic shock plus trau- matic brain injury 7–9 and hemorrhagic shock plus sepsis. 10–12 While the exact mechanism by which VPA exerts its protective effects is unknown, VPA has been shown to regulate a number of proteins and pathways related to cell death, including heat shock protein 70, 13 Hypoxia-inducible factor 1-α (HIF1α), 14 RAC-α serine/threonine-protein kinase (Akt) and glycogen syn- thase kinase-3 β (GSK3β), 6,15 apoptosis regulator Bcl-2 (Bcl-2), 6 β-catenin, 6 brain-derived neurotrophic factor, 16 mitogen-activated protein kinases (ERK/JNK), 15,17 and nuclear factor kB. 17 In prior animal studies of lethal insults, VPA has been given at relatively high doses (150–400 mg/kg), which are six- fold to eightfold higher than what is currently approved by the FDA for clinical use. In human cancer research, the maximum single tolerated dose of intravenous VPA was found to be 60 mg/kg per day. 18 To meet large dose requirements, VPA may be given in divided doses over multiple days. This is not Submitted: January 21, 2016, Revised: June 19, 2016, Accepted: July 8, 2016, Pub- lished online: September 3, 2016. From the Department of Surgery, University of Michigan, Ann Arbor, MI (P.E.G., I.H., V.N., T.B., C.T., B.L., T.L., H.B.A.); Department of Biological Chemistry, Univer- sity of Michigan, Ann Arbor, MI (H.R.); and Department of Computational Med- icine & Bioinformatics, University of Michigan, Ann Arbor, MI (G.H., R.M.). This study was funded by a research grant from the Office of Naval Research (N000141310071) to H.B.A., National Center for Advancing Translational Sciences of the National Institutes of Health under award number UL1TR000433 to the Michigan Institute for Clinical & Health Research. This study was presented at the 46th annual meeting of the Western Trauma Association, February 28–March 4, 2016, in Lake Tahoe, California. Supplemental digital content is available for this article. Direct URL citations appear in the printed text, and links to the digital files are provided in the HTML text of this article on the journal’s Web site (www.jtrauma.com). Address for reprints: Hasan B. Alam, MD, University of Michigan Hospital, 2920 Taubman Center/5331, University of Michigan Hospital, 1500 E. Medical Center Dr., Ann Arbor, MI 48109; email: alamh@med.umich.edu. DOI: 10.1097/TA.0000000000001249 WTA 2016 PLENARY P APER 1020 J Trauma Acute Care Surg Volume 81, Number 6 Copyright © 2016 Wolters Kluwer Health, Inc. All rights reserved.